In cochlear inner hair cells (IHCs), Cav1.3 L-type voltage-gated Ca2+ channels mediate Ca2+ signals that trigger exocytosis of neurotransmitter from IHCs onto auditory nerve afferents. This function of Cav1.3 is crucial for hearing: loss-of-function alterations in Cav1.3 cause deafness in humans and animal models. Cav1.3 channels exhibit distinct properties in IHCs compared to other cell-types, but little is known about what underlies these differences or their relevance for sound encoding by the IHCs. Filling this gap in knowledge is expected to reveal fundamental processes that are required for the unique role of Cav1.3 channels at this first synapse in the auditory pathway. The long-term goal of our research is to define the mechanisms that regulate voltage- gated Cav Ca2+ channels in order to discover what causes, and how to cure, human disease. To this end, we have identified new forms of Cav1.3 modulation in IHCs. First, we found that the cell-surface density of Cav1.3 channels in IHCs is controlled by interactions with harmonin, a protein implicated in the pathogenesis of Usher syndrome. Harmonin enhances degradation of Cav1.3 by the ubiquitin-proteosome (UPS) system, and this process is disrupted in a mouse model of Usher syndrome. Second, we discovered that CaBP2, a Ca2+ binding related to calmodulin (CaM), inhibits Ca2+-dependent inactivation of Cav1.3; this effect is impaired by a human mutation in the CaBP2 gene that causes autosomal-recessive hearing loss. Third, we found that Cav1.3 associates with RIBEYE, the major component of ribbon synapses in IHCs and other sensory cell-types. This interaction may regulate not only the localization, but also the function of Cav1.3 at the IHC active zone. Based on our findings, we hypothesize that the macromolecular assembly of Cav1.3 with proteins such as harmonin, CaBP2, and RIBEYE, dictate the strength and localization of Ca2+ signals in IHCs, and is therefore crucial for auditory transmission. The objective of this proposal is to test this hypothesis using molecular, genetic, and electrophysiological techniques. The rationale is that the proposed research will reveal essential signaling complexes that shape the synaptic function of IHCs, and how dysregulation of such complexes may contribute to the pathophysiology of inherited or acquired forms of hearing loss.